The present invention is directed to methods and reagents which are useful in the determination of hemoglobin and leukocytes in whole blood samples. More particularly, the present invention relates to cyanide-free reagents for use in the rapid formation of a stable and detectable chromogen which is indicative of the amount of hemoglobin present in a whole blood sample by conventional methodology. In addition, the present invention is suitable for leukocyte determinations as well as for hemoglobin measurements. The reagents and methods are particularly suited to application to automated hematology instruments, especially instruments which utilize the same aliquot of whole blood sample with a lyse reagent adapted for both hemoglobin and leukocyte determinations.
The reference or standard methods for determining white blood cell (WBC) counts and hemoglobin (Hb) determinations traditionally utilize reagents containing potassium cyanide (KCN) or other cyanide containing compounds. These cyanide compounds can be hazardous to use because they can produce toxic hydrogen cyanide (HCN). These compounds can also be difficult to dispose of due to the environmentally toxic cyanide content.
Therefore, investigators have been working to develop alternative reagents, which do not contain cyanide. These efforts, however, have certain limitations for application in high-throughput automated instruments which process large numbers of samples, or in a slower methods performed manually or by semi-automated instruments. The chromogen compounds produced by some of these reagents may be formed too slowly in the absence of cyanide or the chromogen produced may be unstable or inconsistent during the period of testing. Additionally, these alternative reagents may not be capable of performing Hb and WBC determinations with the same whole blood sample without making modifications to the instrument hardware or software.
There is, therefore, a great need for a method and reagent system which is free of cyanide and yet reliable for the in vitro analysis of blood utilized by currently available hematology instruments.
The throughput of current automated hematology instruments requires the use of methods and reagents exhibiting rapid reactions rates. For example, the hemoglobin sample turnover rate of an Abbott Laboratories Cell-Dyn(copyright) 3000 instrument is approximately twelve (12) seconds. Abbott Laboratories Cell-Dyn(copyright) 1600 and 3500 instrument systems take approximately twenty-four (24) seconds for hemoglobin determinations. In addition, most of these instruments call for the same blood sample aliquot and reagent systems to be used for both the white blood cell size and population determinations, as well as the concentration of hemoglobin in the erythrocytes. Current hematology analyzers often utilize the same dilution and reaction mixtures of whole blood and reagent for both hemoglobin determination and white blood cell counting and sizing.
The xe2x80x9cstandardxe2x80x9d lysing/hemoglobin reagent typically contains ingredients to properly lyse the erythrocytes to permit accurate leukocyte counting, and a cyanide containing compound for the formation of a stable chromogen (cyanmethemoglobin) to enable precise colorometric analysis of the hemoglobin content in the erythrocytes. Therefore, in order for a new lysing reagent to be practical, it must be easily adopted for use in existing automated and semi-automated hematology instruments without alteration of either the instrumentation or the performed methodologies. Consequently, there are many significant requirements which must be met by a cyanide-free lysing reagent. Some of these are:
1. The chromogen produced should have maximum absorbance between 530 nm and 550 nm, which is the optimal absorbance range for cyanmethemoglobin of the majority of current, automated methodologies.
2. The chromogen produced should not only be quick to form, but also be fairly stable for a period of at least five minutes to provide good, reproducible results in automated or semi-automatic methods of hemoglobin and leukocyte determinations.
3. The lysing reagent should not interfere with the formation of the hemoglobin chromogen and cannot adversely affect the leukocytes"" stability during leukocyte sizing and counting procedures.
Methods are provided for making Hb and WBC determinations and compositions comprising diluting a whole blood sample with a diluent and mixing the whole blood sample/diluent with an aqueous lyse reagent comprising from about 0.1 to about 20% by weight of at least one quartenary ammonium salt selected from the group consisting of: tetradecyltrimethyl ammonium bromide (TTAB), dodecyltrimethyl ammonium chloride, cetyltrimethyl ammonium bromide, hexadecyltrimethyl ammonium bromide, benzalkonium chloride, cetylpyridium chloride and from about 0.1 to about 15% weight of hydroxylamine salts selected from the group consisting of: hydrochloride, sulfate, phosphate and other acid salts. A chromogen is formed, detected and measured thereby indicating Hb concentration in the whole blood sample as well as WBC population and subpopulation determinations. In addition, compositions are provided of multipurpose cyanide-free lyse reagents which can be utilized in various instrumentations.